c-Met is a receptor protein tyrosine kinase of the same family as epidermal growth factor (EGF) receptors. This transmembrane protein acts as the cell surface membrane receptor in which the extracellular domain (ECD) binds hepatocyte growth factor/scatter factor (HGF/SF, also abbreviated HGF herein). c-Met, used interchangeably with “Met”, is a disulfide-linked heterodimer with α (Mr 45,000) and β (Mr 145,000) subunits. The α chain is located outside the cell membrane, whereas the β chain consists of an extracellular domain, a single transmembrane domain, and a cytoplasmic moiety in which the receptor tyrosine kinase domain resides (Birchmeier, C., et al. (2003) Nat. Rev. Mol. Cell Biol. 4, 915-925). c-Met dimerizes after binding ligand to form the active kinase. The intracellular tyrosine kinase domain activates a complex cascade of biochemical reactions. The c-Met receptor kinase regulates cellular proliferation, migration, differentiation and branching morphogenesis during development and homeostasis.
c-Met is also expressed on the cell surface of a variety of human primary solid tumors and in their metastases (http://www.vai.org/met/). In its activated state, the c-Met receptor controls growth, invasion, and metastasis of cancer cells through multiple signal transduction pathways. In some cancer cell lines, loss of c-Met expression through silencing promotes apoptosis, demonstrating that c-Met is necessary for survival. Met activity increases through mutations in the kinase or juxtamembrane domains, through overexpression, or through binding to HGF. In addition to increased c-Met expression, elevated HGF/SF concentrations in the tumor microenvironment have also been associated with adverse outcome. For example hypoxic tumor stromal cells in pancreatic cancer increase HGF secretion and accelerate pancreatic cancer progression.
c-Met is one of the most frequently genetically altered or otherwise dysregulated receptor tyrosine kinases (RTK) in advanced human cancers and thus represents an attractive treatment target. Kinase activating c-Met mutations are observed in sporadic renal, lung, head and neck, hepatocellular carcinoma, non small cell lung cancer (NSCLC), gastric cancer and melanoma. Furthermore, amplification of the c-Met locus has been detected in gastric, metastatic colorectal and esophageal adenocarcinoma, additional c-Met-related diseases. Activation of c-Met in cancer cells induces the secretion of angiogenic factors, such as VEGFA and IL-8 and inhibits synthesis of thrombospondin-1, an anti-angiogenic factor. In addition, c-Met activation in endothelial cells causes angiogenesis. While the cytotoxic effects of inhibiting Met activity may only occur in cancers with activated c-Met, the antiangiogenic effect may exist more frequently. Any disease associated with Met expression is referred to herein as a “Met-related disease”.
The magnitude of c-Met expression predicts the aggressiveness of a number of cancer types (http://www.vai.org/met/). Accurate detection and quantification of c-Met protein expression are needed to identify cancers that are likely responsive to c-Met inhibitors and the development of such molecular diagnostics lags significantly behind the drug development.
Antibody molecules and their derivatives have tremendous potential for use in a variety of research, diagnostic, and therapeutic applications. To provide targeting specificity, it is useful to develop a targeted cancer therapeutic by generating a ligand that specifically binds to a receptor which is tumor specific or sufficiently over-expressed in the tumor (Scheffer, et al. (2002) Br J Cancer, 86:954-962; Gura, T. (2002) Nature, 417: 584-586; Popkov, M., et al. (2005) Cancer Res. 65: 972-81; Wilson, I. A. et al. (1994). Curr. Opin. Struct. Biol. 4, 857-867; Weiner, L. et al. (2005) Nature Biotech 23, 556-557). Antibodies have proven to be important targeting ligands for cell surface receptors. Recent progress in the manipulation of antibody subunits using molecular techniques, plus the ability to reproducibly select an antibody, an antibody fragment, or a peptide, allows the generation of useful binding subunits, while avoiding many of the problems that have often been associated with these molecules.
All antibodies are immunoglobulin (Ig) molecules made up of two heavy (H) and two light (L) chains. Each H and L chain are linked by a disulfide bridge which is just N-terminal of a “hinge” region. Pairs of two chains, disulfide linked, include the same basic unit of four polypeptide chains: two light chains (L) and two heavy chains (H). Both the heavy chains and light chains have intra-chain disulfide bridges, which create polypeptide loops or domains, of about 110 amino acids. These domains are referred to as VH (variable domain of the H chain), VL (variable domain of the L chain), CH1, CH2, CH3 (constant domains of the H chain), and CL (constant domain of the L chain). An Fab fragment was originally defined as is a papain digestion product of an intact antibody made of the N-terminal “half” of the H-chain which is now defined as VH-CH1 and all of the L-chain which is now defined as VL-CL. The Fab fragment contains the antigen binding site defined by the VH and VL domains of the H and L chains, respectively. Hence, the antigen binding site of any antibody (or antigen binding fragment thereof) is made up of the V domains (VH and VL) that interact physically with one another. When physically associated, these domains together are also referred to as an Fv fragment, while recombinant forms of these domains in the form of a single chain are referred to as single chain Fv fragments (scFv).
The term “CDR” refers to the complementarity determining region or hypervariable region amino acid residues of an antibody which are responsible for antigen-binding. Framework or “FR” amino acid residues are those variable domain residues other than and bracketing the CDR regions. The variable (V) domain of an Ig chain includes hypervariable (HV) regions which are also known as complementarity-determining regions (CDRs) because they are important in “determining” the structure of the antibody combining site that is complementary the epitope bound. Each H and L chain V region has three HVs or CDRs. The segments on either side of each HV region which are relatively invariant are termed “framework regions” (FRs). Thus, the order of these regions in a V domain (from the N-terminus) is as follows: FR1-HV1-FR2-HV2-FR3-HV3-FR4. For example, the three HV regions are roughly from residues 28-35, 49-59 and 92-103, respectively. The framework regions form the β-sheets that provide the structural framework of the domain, with the HV sequences corresponding to three loops at one edge of each sheet that are juxtaposed in the folded protein. The HV loops from the VH and VL domains are brought together, creating a single HV site at the tip of the Fab fragment which forms the antigen binding site. (See, for example, Janeway, C. A., Jr. et al., IMMUNOBIOLOGY, 2n ed., Garland Publishing Inc., New York, 1996, chapter 3).
One antigen validated for raising antibodies is c-Met. Mouse mAbs against human c-Met and against human HGF/SF have been generated (Ohashi, K., et al. (2000) Nat. Med. 6, 327-331; Cao, B., et al. (2001) Proc. Natl. Acad. Sci. USA 98, 7443-7448; Nguyen, T. H., et al. (2003) Cancer Gene Ther. 10, 840-849), and some of them showed strong affinities both in vitro and in animal models. However, clinical problems resulting from the formation of human antimurine antibodies and other pharmacodynamic effects have hampered their efficacy in the human body.
The mechanism by which antibodies produce therapeutic outcomes include antibody dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity, and the blocking of signal pathways that promote uncontrolled cell proliferation, e.g., with a neutralizing antibody (Von Mehren M, et al. Annu Rev Med 54:343-69 (2003)).
Antibodies are good delivery vehicles for targeting agents for treatment or in vivo diagnosis. Antibodies can be conjugated to a compound (e.g., a nucleic acid, chemotherapy compound, toxin, or radionuclide) to direct such compound to cancer cells by recognizing the specific tumor marker on the cell surface. Antibody-conjugated drug, consisting of antibodies coupled to toxins or chemicals, are extremely useful tools in the elimination of specific cell populations in vitro and in vivo for research and therapeutic applications. The antibody is used to target the drug to a specific cell population, which is distinguished by its cell-surface antigen (Groner, B., et al. (2004), Current Molecular Medicine 4, 539-547; Watkins N A, et al. (2003) Blood. 102:718-24; Rousselet N, et al. (2004) Cancer Res. 64: 146-51). This can be done by conjugating chemotherapeutic drugs, radionuclides, or immunoliposomes for nanoscale delivery, and by directly fusing the antibody to an immunotoxin or cytokines using recombinant technology (Wu A M, et al. Nature Biotech 23:1137-46 (2005); Mamot C, et al. Cancer Res 65:11631-38 (2005)). An antibody-conjugated drug delivery system can direct toxic compounds to penetrate the targeted malignant tissue or cells specifically, thus decreasing the toxicity of drug to normal cells nearby and elsewhere, and increase the therapeutic effect by enhancing the accumulated dose of the drug in the targeted tissue. Antibody conjugates eliminate damage that otherwise would be caused to normal cells if such compounds were delivered to a patient by traditional chemotherapy regimens (Schrama D, et al. (2006) Nature Reviews of Drug Discovery 5: 147-159).
Beside the conjugation of chemotherapeutics, radionuclides or toxins, antibody conjugates have been developed in many fields, including the antibody-cytokine fusion protein for immune modulation treatment, antibody-ligand fusion protein for apoptosis induction, and the antibody directed enzyme pro-drug targeting (ADEPT).
Conjugation also increases the solubility of some compounds in physiological solutions, enhancing the stability of the compounds, and preventing the conjugated drug from being pumped out of cells by the multidrug resistance associated p-glycoprotein transmembrane pump (Guillemard V, et al. Cancer Res 61: 694-9 (2001); Guillemard H, et al. Oncogene 23:3613-21 (2004)).
When using an antibody for drug delivery, internalization of the antibody is generally desired since it allows some compounds to be delivered intracellularly, for example after release, from the antibody. For the purpose of chemoimmunoconjugation, an antibody that can be endocytosed by a cell is essential since an internalized antibody can lead the conjugated molecules into the cells to accumulate. Some linkers of the conjugation also need to be internalized to release the compounds by the lysosome (Schrama D, et al. (2006) Nature Reviews of Drug Discovery 5: 147-159).
An antibody conjugated to a detectable label such as a radionuclide provides a method for early diagnosis of a malignancy, when a cell surface protein is overexpressed or mutated so that it is distinguishable by an antibody from the unmutated form. Established methods for radiolabeling mAbs in suitable quantity and of appropriate quantity for scintigraphy are available, feasible, relatively inexpensive, and adaptable to virtually any mAb regardless of its epitopic specificity. New radiolabeling methods are continually emerging, and many laboratories are evaluating a wide range of antibody derivatives—from full-length chimeric and humanized molecules, to monomeric and multimeric antibody fragments, to immunoconjugates—as potentially superior imaging and therapeutic agents, with improved targeting selectivity and more favorable biological turnover kinetics (Program and Abstracts, Ninth Conference on Cancer Therapy with Antibodies and Immunoconjugates. 2002. Cancer Biotherapy & Radiopharmaceuticals 17:465-494).
Moreover, the reagents, supplies, and equipment required to perform radioimmunoscintigraphy in experimental animals and in humans are commonplace. For decades decommissioned or refurbished clinical gamma cameras have proven satisfactory for animal imaging applications, and they continue to do so. Modified or custom-built gamma cameras adapted for small animal imaging are becoming more widely available.
The major advantage of scintigraphy as a molecular imaging modality (not limited to imaging with antibodies) is that the acquired images are inherently quantitative. The physics of gamma radiation and the mathematical analysis of nuclear images, including corrections for photon attenuation and other artifacts, are well understood. In animal models, as well as in human studies, one can noninvasively and accurately measure net accumulation and some kinetic parameters of radiopharmaceutical interactions with target lesions, and the concurrent collection of even a small set of biological samples (e.g., blood and excreta) for direct counting combined with quantitative analysis of diagnostic images enables one to make useful dosimetry estimates for therapeutic purposes.
Many different radiopharmaceuticals are available for imaging neoplasms. They range from classical agents such as sodium iodide (Na131I, thallium chloride (201TlCl), and gallium citrate (67Ga-citrate) to highly selective positron-emitting reporter gene detection systems (Vallabhajosula S (2001) Nuclear Oncology; I Khalkhali et al., eds. Lippincott Williams & Wilkins, Philadelphia, Pa. pp. 31-62; Iyer M et al. (2001) J Nucl Med 42, 96-105). Radiolabeled molecules that bind to specific cell surface components provide one successful approach to tumor imaging and therapy. Examples are OCTREOSCAN® for imaging and potentially treating neuroendocrine neoplasms, CEASCAN® and ONCOSCINT® for imaging colorectal and ovarian cancers, and BEXXAR® and ZEVALIN® for detecting and treating certain lymphomas.
Relatively smaller antibody fragments have higher penetrating speed into the solid tumor tissue (Holliger P, et al. Nature Biotechnology 23: 1126-36 (2005)), plus, if they are xenogeneic to the host, they have fewer foreign epitopes that can be recognized by the recipient's immune system. To avoid such immunogenic effects of xenoantibodies, most antibodies in clinical trials or use are human antibodies or at least chimeric (and humanized) antibodies so that they comprise a human constant regions linked to the original, xenogeneic (typically murine) variable regions.
Not all antibodies are suitable for creating a therapeutic drug, and large numbers of antibodies may need to be screened. This is a time-consuming and expensive process if each potential candidate must be conjugated to the drug and purified (Hudson P J, et al. (2003) Nat Med. 9:129-34; Kohls, M. et al., (2000) Biotechniques, 28: 162-165.
Since the 1970s, rodent antibodies have been widely applied for medical purposes, and mainly for in vitro diagnosis (Kohler G, et al (1975 Nature 256:495-497). The first therapeutic murine mAbs clinical study was performed in the early 1980s, but failed due to human anti-mouse antibodies (HAMA), short serum half-lives, and low efficacy of interaction with human immune effector cells (Reichert J M, et al (2005) Nature Biotechnology 23:1073-1078). However, completely human antibody fragments can be used to minimize the regions of non-human origin and thereby increase clinical tolerance. (Liu, Y., et al (2004) Int J Cancer 108, 549-557; Adams, G., et (2001) Cancer Res. 61: 4750-4755; Souriau, C. et al. (2004) Growth Factor, 22(3):185-194; Lui, V. W. et al (2002) Anticancer Res. 22, 1-11).
Human antibodies are desired for treatment and in vivo diagnosis of patients in order to prevent the HAMA response that could eliminate non-human antibodies and thus decrease the effect of the non-human antibodies after the first use. The development of recombinant technologies make it possible to generate a chimeric antibody (combining the variable region of a mouse and the constant region of human) or a fully human antibody for clinical use. Currently, three kinds of technologies have been developed to make a fully human antibody: a fully human antibody raised by a transgenic mouse (Jakobovits A (1995) Curr Opin Biotechnol 6: 561-566), humanization of a murine antibody (Jones P T, et al (1986) Nature 321: 522-525), and panning of recombinant human antibody libraries.
There are numerous reports of human antibodies successfully raised from combinatorial libraries that are representative of the natural immune response (Huls, G. et (2001) Intl Cancer Res. 50, 163-171; Huls, G A, et al (1999) Nat Biotechnol. 17:276-81; Begent R H, et al. (1996) Nat Med. 2:979-984; Liu, B., et al. (2004) Cancer Res. 64: 704-710; Scheffer G L, et al. (2002) Br J Cancar, 86:954-962; Hudson P J, et al. (2003) Nat Med. 9:129-34).
Using phages to display non-immunized libraries consisting of large numbers of IgG fragments, Fab fragments or scFvs for specific antigens have been recovered. But the antibodies fragments from these libraries often have weak affinities (in the range of 10−6 M). Phage display of antibody combinatorial libraries not only creates the direct physical linkage between genotype and phenotype that exists for B lymphocytes, but can also mimic in vitro many of the in vivo processes which result in the production of high-affinity Abs (Huls, G, et al. (2001) Intl Cancer Res. 50, 163-171; Huls G A, et al. (1999) Nat Biotechnol. 17:276-81).
Because c-Met is overexpressed in solid tumors, c-Met is an ideal target for antibody-directed drug delivery and for tumor imaging. To achieve this aim, a human anti-Met antibody, antibody Fab fragment, or scFv fragment is desired.